Dietary polyphenols identified as intracellular protein kinase A inhibitors.
BACKGROUND
Dietary plants contain several thousands different polyphenols that can potentially influence normal and pathological cellular processes through modulation of intracellular signaling pathways. A few polyphenols have been shown to be potent inhibitors of protein kinases.


OBJECTIVE
To identify possible dietary protein kinase A (PKA) inhibitors we designed a method for screening of substances in crude mixtures of food items for modulation of intracellular PKA activity that enables high-throughput testing of a large number of compounds and extracts.


METHODS
Luciferase was mutated to render it sensitive to phosphorylation by PKA (luciferase(PKA)) and transfected into a human hepatoma cell line (HepG2). Cells were then treated with extracts from dietary plants, including berries, fruits and spices, and intracellular PKA-activity was assessed by change in bioluminescence in live cells by imaging.


RESULTS
Several extracts were found to inhibit PKA activity in a 96-well platform high-throughput screen. Green tea, crowberry, clove and cinnamon extracts were found to reduce intracellular cAMP levels consistent with their ability to increase luminescence from luciferase(PKA). Also pomegranate extract inhibited intracellular PKA and was used to estimate cellular association of polyphenols by HPLC and LC-MS. Pomegranate extract contains several anthocyanins, including delphinidin-3 glucoside. Delphinidin aglycone was found to inhibit cellular PKA activity in a concentration dependent manner. The inhibitory activity was found to be structure specific as a closely related compound to delphinidin had no activity.


CONCLUSIONS
The current work identify phytochemicals in crude extracts which modulate cell signaling through PKA in a way that facilitate high through-put screening to help elucidate how plant based diet reduce risks of chronic diseases.
Moskaug
Jan Øivind
JØ
Dept of Biochemistry, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway. j.o.moskaug@medisin.uio.no
Borge
Grethe I
GI
Fagervoll
Anne M
AM
Paur
Ingvild
I
Carlsen
Harald
H
Blomhoff
Rune
R
D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D005419:Flavonoids / Q000494:pharmacology*; D006801:Humans; D008156:Luciferases / Q000378:metabolism*; D049449:Luminescence; D008163:Luminescent Measurements; D013058:Mass Spectrometry; D010636:Phenols / Q000494:pharmacology*; D059808:Polyphenols; D015398:Signal Transduction; D014162:Transfection
D005419:Flavonoids; D010636:Phenols; D059808:Polyphenols; D008156:Luciferases; D017868:Cyclic AMP-Dependent Protein Kinases